Alcoholism and alcohol use disorders (AUDs), which are characterized by repeated episodes of binge drinking, contribute to health problems in the US veteran population. Binge drinking, defined as drinking that brings blood alcohol concentration to 80 mg% or above, appears to be a persistent problem in the military, and it is prevalent in young men entering the military. It has been hypothesized that the experience of stress may represent a risk factor that shifts alcohol consumption from recreational to excessive. We contend that prior binge drinking is another risk factor that interacts with stress to augment subsequent alcohol drinking behavior. During the current funding period, we developed an animal model where prior binge alcohol (ethanol) experience and repeated exposures to predator odor stress (considered a traumatic stress and used as a model of post-traumatic stress disorder, PTSD), significantly increased subsequent ethanol intake in C57BL/6J male mice. The proposed studies will use this model to test the hypothesis that the combination of binge ethanol intake and exposure to traumatic stress produces behavioral and molecular adaptations that confer susceptibility for increased ethanol intake in male mice. Based on evidence that a high anxiety state and/or changes in ethanol's rewarding and aversive properties might contribute to augmented ethanol intake, Aim 1 will determine whether the synergistic effect of traumatic stress and binge drinking to enhance later ethanol intake is associated with alterations in anxiety, ethanol reward, or ethanol aversion. Four groups of C57BL/6J male mice will be tested. Groups 1 & 2 will have 7 binge ethanol sessions or consume water, respectively. After 1 month of abstinence, 23 hr ethanol drinking will be measured for 4 weeks (1 week baseline, 2 weeks with intermittent predator odor stress, 1 week post-stress). Group 3 will be exposed to intermittent predator odor stress only, and Group 4 is the nave control. At 1 week following the post-stress drinking or the final predator odor stress, mice will be tested for anxiety (elevated plus maze), ethanol reward (conditioned place preference), and ethanol aversion (conditioned taste aversion). We predict that enhanced ethanol intake following prior binge drinking and traumatic stress will be associated with an increase in anxiety and an increase in ethanol's rewarding properties. Aim 2A will use customized neuroscience mouse qPCR arrays to characterize gene expression changes in the nucleus accumbens (NAc) to identify novel signaling pathways and determine common and distinct neuro-adaptive networks regulated by traumatic stress or the combination of binge drinking and traumatic stress. During the current funding period, we used qPCR arrays to identify and subsequently confirm changes in several signaling cascades in the NAc at 24 hr following 7 ethanol binges. To build on these findings, mice will be treated as in Aim 1, but will be euthanized at 24 hr following the post-stress drinking or final predator odor stress. We predict that NAc gene expression will be differentially altered by traumatic stress or binge drinking and that further novel adaptations will occur with the combination of traumatic stress and binge drinking that may mediate the synergistic effect to increase drinking. Aim 2B will conduct biological confirmation of select target genes that are involved in the expression of enhanced ethanol intake, using strategies that we successfully employed with current funding (e.g., Westerns and phospho-Westerns, measurement of corticosterone and neurosteroid levels, or manipulate molecules pharmacologically and determine the effect on increased ethanol intake). Information gained from the multidisciplinary approach will further elucidate the importance of binge drinking and traumatic stress as risk factors for AUDs and guide treatment strategies to reduce AUDs. Given the number of young adults with binge drinking experience now serving in the military and undergoing multiple deployments and the comorbidity of PTSD and alcoholism in veterans, the proposed studies are timely and very important.